SUMMARY Development of Fluorescent False Neurotransmitters Monoamine neurotransmission plays important roles in modulation of excitatory and inhibitory synapses in the central nervous system and thus provides essential fine tuning of behavior in response to a changing environment. Consequently, aberrations in monoamine neurotransmission underlie many neurological and neuropsychiatric disorders including Parkinson?s disease, schizophrenia, ADHD, depression, and drug addiction. We have established an interdisciplinary research program focused on development of new molecular probes to study monoamine neurotransmission on a synaptic level. Specifically, we have introduced a conceptually new class of probes, termed ?fluorescent false neurotransmitters? (FFNs), that act as optical tracers of dopamine and norepinephrine. FFNs provide the first experimental means to image synaptic vesicle content uptake and release at individual presynaptic varicosities in brain tissue and in living animals. Using FFNs and multiphoton microscopy imaging we have made several unexpected findings, including the discovery of silent dopaminergic and noradrenergic synapses, activity-dependent super-charging of synaptic vesicles, and mechanistic insights on psychostimulant drugs. In this renewal application, we propose to expand the scope of FFNs to serotonin synapses, the third major monoamine system, on the basis of proof-of-concept studies. We will also develop new imaging and photon-counting methods based on the combinatorial use of FFNs as presynaptic probes with emerging genetically encoded monoamine reporters as postsynaptic sensors to provide multi-parameter synaptic readouts in living mice. In addition to complementing each FFN class with the corresponding sensor (for example, dopamine FFN and dopamine sensor), mixing and matching FFNs and sensors each for a different monoamine will enable simultaneous examination of two neurotransmitter systems. This in turn will enable the study of complex processes such as co-transmission and neurotransmitter switching on a synaptic level. We will also examine the effect of monoamine releasing agents on synaptic neurotransmitter dynamics at the dopamine, norepinephrine, and serotonin axonal release sites to probe both the vesicular pools in active and silent varicosities and the mechanisms of action of these psychoactive drugs. This application addresses a major unmet need in developing molecular tools and methods for imaging multiple neurotransmitter systems in living animals with adequate spatial and temporal resolution.